Sodium transporting epithelia, such as renal distal and collecyting tubules, function to control whole-body homeostasis. Epithelial sodium channels (ENaC) have been found in other sodium transportinmg epithelia, e.g., salivary glands, colon, bronchial and tracheal epithelia, as well as in many non-epithelial cells, like lymphocytes, neurons and astrocytes. Amiloride inhibition is a hallmark of these particular channels, regardless of the system in which they are found. Yet, when macroscopic and single channel properties are examined using the patch clamp technique, a myriad of biophysical characteristics emerge. The central hypothesis of this application is that the observed functional diversitiy of amiloride-sensitive sodium channels results, in part, from different combinations of subunits of the Degenerin(DEG)/ENaC superfamily of ion channels. There are three specific aims. In the first specific aim, we will detrermine the biochemical composition of an amiloride-sensitive cation channel found in epithelial cells that exhibit biophysical properties different from ENaC. We present preliminary data using RT-PCRprofiling showing that message for a variety of DEG/ENaC memebers are present in these cells. Thus, we will use these cells as ,model systems to determine the biochemical composition of this channel. In addition, we will employ surface biotinylation, surface chemiluminescence, co-immunoprecipitation analysis, and macromolecular assembly assays, to verify subunit interactions. We will also use ellular protein knockout approaches, (and MTS reagent susceptibility studies), to establish subunit interaction. The second specific aim is to: a.) determine the biophysical characteristics of hybrid ENaC/ASIC, b.) identify ENaC/ASIC interaction using MTS reagent susceptibility. The third specific aim will determine the high resolution crystal structure of ?ENaC. These results wil offer new insights into the nature and ultimately the regulation of amiloride-sensitive sodium channels, and the ways that these hybrid channels can be modulated by inserting or deleting specific subunits of this DEG/ENaC superfamily. Thus understanding the molecular basis for ENaC diversity will provide unique opportunities for therapeutic interventions in an ever-increasing plethora of EWNaC-related diseases. PUBLIC HEALTH RELEVANCE: Epithelial sodium channel (ENaC) proteins and acid-sensing ion channel (ASIC) proteins are distributed all over the human body, are responsible for maintaining the body's salt and water balance, and play a role in hypertension, cancer, learning, and many other normal and abnormal processes. Through modern techniques of molecular biology, biochemistry, cell biology, and electrophysiology, our study results will provide insight into the cellular mechanisms of these channels in native tissues, especially in renal epithelia. The relevance of this particular study extends to autosomal recessive polycystic kidney disease, which affects 1 in 20,000 babies in the United States.